This project will define the essential mechanics of macropinosome closure. Macropinocytosis and morphologically analogous movements of membrane and the actin cytoskeleton are important in epithelial physiology and host defense against many infections. The successful completion of macropinocytosis always requires closure: the constriction of a region of the cell surface that transforms a cup-shaped invagination of plasma membrane into a discrete membrane-bounded organelle inside the cell. Despite its ubiquity and importance in health and disease, the mechanics and regulation of closure remain largely unexplained. Morphology predicts two distinct stages of macropinosome closure: constriction at the distal margin of cup-shaped, cell surface ruffles, which requires phosphoinositide 3'- kinase (PI3K), followed by a scission that separates the macropinosome from the plasma membrane. The macropinocytic cup can be considered as a transient, self-organized contractile structure at the cell surface. We hypothesize that PI3K organizes actin-myosin-based contractile activities localized to the inner membrane of the macropinocytic cup, which are maintained by a barrier at the distal rim that restricts lateral diffusion of 3'phosphoinositides out of the cup. This hypothesis will be tested by identifying precisely the point of macropinosome closure in macrophages and by localizing barriers to diffusion at various stages of macropinosome formation. Specific Aim 1 will identify the timing and morphology of closure. The point of scission will be identified by measuring access to macropinosomes of externally added, membrane-impermeant fluorescent probes. Instruments and methods developed in this lab will be used to acquire time-resolved, 3-dimensional (4D) reconstructions of constriction and scission in cells expressing fluorescent probes. Specific Aim 2 will identify barriers to lateral diffusion in macropinocytic cups. Morphological studies suggest that during closure the rim of the cup prevents diffusion of inner-leaflet phospholipids out of the cup. A barrier to diffusion at the cup rim could facilitate the local accumulation of 3'phosphoinositides and constrain contractile activities to the inner membrane of the cup. To identify such barriers, fluorescent chimeras of photoactivatable GFP (paGFP) with membrane-anchoring protein domains (paGFP-MEM) will be locally photoactivated in or near macropinosomes or cups. If diffusion of paGFP-MEM in the plane of the bilayer is restricted by cup rims, then fluorescent paGFP-MEM generated inside cups should be constrained to the cup domain of the plasma membrane. Concentration gradients of phosphoinositides within cups and across the cup rim will be measured by quantitative microscopy of cells expressing fluorescent, phospholipid-binding domains. Thus, by defining the spatiotemporal organization of cytoplasm during closure, these studies will create a framework for analyzing molecular mechanisms of closure in macropinosomes and phagosomes. PUBLIC HEALTH RELEVANCE: Pinocytosis is a microscopic drinking activity, present in nearly all living cells, which is especially important in immunity and in kidney function. This work will analyze an unexplained but essential last step in the process, in which an invagination of the cell's plasma membrane closes to form a fluid-filled vesicle inside the cell. The results should have implications for how cells coordinate large-scale activities in their cytoplasm.